Filipp Dominik, Leung Bernadine L, Zhang Jenny, Veillette André, Julius Michael
Sunnybrook and Women's College Health Sciences Center and Department of Immunology, University of Toronto, Toronto, Ontario, Canada.
J Immunol. 2004 Apr 1;172(7):4266-74. doi: 10.4049/jimmunol.172.7.4266.
Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCRbeta chain. The present study uses a family of murine IL-2-dependent CD4(+) T cell clonal variants in which anti-TCRCbeta signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag- and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4-associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRCbeta responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCRzeta:zeta-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4(+) T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRCbeta signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRCbeta proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.
近期的研究结果深入揭示了调控依赖lck的fyn激活的时空关系，并证明了TCR/CD4诱导的lck激活和易位至脂筏以及随后共定位的fyn的激活。由此预测，将lck直接靶向脂筏将绕过TCR和CD4-lck并列的需求，并挽救由针对TCRβ链恒定区的抗体介导的细胞激活。本研究使用了一组小鼠IL-2依赖的CD4(+) T细胞克隆变体，其中抗TCRCβ信号以lck依赖的方式受损。重要的是，这些变体对Ag和mAb介导的TCR-CD4共聚集有反应，这两者都能使与CD4相关的lck与TCR/CD3复合物进行协同相互作用。我们之前已经证明，该系统中抗TCRCβ的反应性与激酶活性、膜相关的lck以及预先形成的低磷酸化TCRζ:ζ相关的70 kDa复合物的存在相关，这种表型在原代静息CD4(+) T细胞中也有重现。我们在本研究中表明，野生型lck的强制表达实现了TCR/CD3复合物相同的基础组成，但并未挽救抗TCRCβ信号。相反，无法结合CD4的C20S/C23S突变的lck（双半胱氨酸lck）的强制表达挽救了抗TCRCβ近端信号和细胞生长。双半胱氨酸lck靶向脂筏，与>98%的细胞fyn共定位，并导致基础fyn激酶活性增加7倍。CD4和TCR的共聚集也能达到相同的结果。这些结果强调了脂筏在空间上协调lck和fyn之间相互作用的关键作用，而这种相互作用是近端TCR/CD3信号传导的前提。
